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Insulin-like growth factor and insulin receptors in intestinal mucosa of neonatal calves I P Georgiev, T M Georgieva, M Pfaffl1, H M Hammon and J W Blum Division of Animal Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing, Faculty of Veterinary Medicine, University of Berne, Bremgartenstr 109a, CH-3012 Berne, Switzerland 1

Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Weihenstephanerberg 3, D-85354 Freising-Weihenstephan, Germany

(Requests for offprints should be addressed to J W Blum; Email: [email protected]) (I P Georgiev and T M Georgieva are currently at Department of Veterinary Physiology and Physiological Chemistry, Faculty of Veterinary Medicine, Thracian University, 6000 Stara Zagora, Bulgaria)

Abstract Intestinal development is modified by age and nutrition, mediated in part by insulin-like growth factors (IGF-I, IGF-II) and insulin. We have investigated whether expression of IGF-I, IGF-II and insulin receptors (IGFIR, IGF-IIR and IR; measured by real-time RT-PCR) and binding capacity (Bmax) of IGF-IR, IGF-IIR and IR in the mucosa of the small and large intestine of neonatal calves are modified by age and different feeding regimes. In experiment 1, pre-term (GrP) and full-term (GrN) calves (after 277 and 290 days of pregnancy respectively) were killed immediately after birth before being fed; a further group of full-term calves were fed for 7 days and killed on day 8 of life (GrC1–3). In experiment 2, full-term calves were killed on day 8 after being fed first-colostrum for 7 days (GrCmax), colostrum of the first six milkings for 3 days (GrC1–3) or milk-based formula for 3 days (GrF1–3). Intestinal sites differed with respect to expression levels of IGF-IR (duodenum>jejunum in GrC1–3; ileum>colon, duodenumdjejunum in GrF1–3), IGF-IIR (colon>duodenum and ileum in GrN), and IR (lowest in ileum in GrP and CrN; highest in colon in GrC1–3 and GrCmax). They also differed with respect to Bmax of IGF-IR (ileum and colon>duodenum and jejunum in GrP; ileum and colon>jejunum in GrN; colon>jejunum in GrC1–3; lowest in jejunum in GrF1–3), IGF-IIR (duodenum and colon>jejunum and ileum in GrP; duodenum>ilem and colon>jejunum in GrN; duodenum, jejunum and

colon>ileum in GrCmax, GrC1–3, and GrF1–3) and IR (ileum>duodenum, jejunum and colon in GrCmax, GrC1–3, and GrF1–3). There were significant differences between groups in the expression of IGF-IR (GrF1–3 > GrCmax and GrC1–3 in ileum), IGF-IIR (GrN>GrP and GrC1–3 in colon; GrN>GrC1–3 in jejunum and total intestine), and IR (GrCmax >GrF1–3 in colon) and in the Bmax of IGF-IR (GrP>GrN in colon; GrCmax >GrF1–3 in jejunum), IGF-IIR (GrN>GrP in duodenum, ileum and total intestine; GrN>GrC1–3 in duodenum, ileum, colon and total intestine) and IR (GrN>GrP in total intestine; GrC1–3 >GrN in ileum and total intestine). In addition, Bmax values of IGF-IR, IGF-IIR and IR were correlated with villus circumference, villus height/crypt depth and proliferation rate of crypt cells at various intestinal sites. There were marked differences in Bmax of IGF-IR, IGF-IIR and IR dependent on mRNA levels, indicating that differences in Bmax were the consequence of differences in posttranslational control and of receptor turnover rates. In conclusion IGF-IR, IGF-IIR and IR expressions and Bmax in intestinal mucosa were different at different intestinal sites and were variably affected by age, but not significantly affected by differences in nutrition. Receptor densities were selectively associated with intestinal mucosa growth.

Introduction

digestive functions are critical due to the change from primarily parenteral nutrition during the fetal stage to exclusively enteral nutrition. Insufficient morphological and functional adaptations of the GIT are considered to be of central etiological importance for GI diseases (Guilloteau et al. 1997). This is especially true for pre-term calves (S Bittrich, H M Hammon and J W Blum, unpublished

In calves, the first 2–3 postnatal weeks are well known to be characterized by high morbidity and mortality rates. Diseases especially of the gastrointestinal (GI) tract (GIT), such as severe diarrhea, are frequent and are often followed by fatal systemic diseases. Adaptations of the GIT and of its

Journal of Endocrinology (2003) 176, 121–132

Journal of Endocrinology (2003) 176, 121–132 0022–0795/03/0176–121 2003 Society for Endocrinology Printed in Great Britain

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observations). Better knowledge of factors that stimulate functional development of the GIT for adequate adaptation to the postnatal period is expected to reduce digestive disorders and to improve digestive efficiency, health status, well-being and growth performance. Morphology and function of the GIT in the postnatal period are influenced by endogenous and exogenous factors. Among exogenous factors nutritional and nonnutritional (bioactive) factors that are ingested with colostrum play a major role, as has been shown in calves by several authors (Guilloteau et al. 1997, Bühler et al. 1998, Blättler et al. 2001, Blum & Baumrucker 2002, Blum et al. 2002). Colostrum at the onset of lactation contains the greatest amount of bioactive substances, including growth factors and hormones to which the newborn calf – and primarily the GIT – is exposed (Blum & Baumrucker 2002). The somatotropic axis of neonatal calves is basically functioning, although it is not yet fully mature (Hammon & Blum 1997). During the postnatal period insulin-like growth factor (IGF)-I becomes more important than IGF-II (Breier et al. 2000, Butler & LeRoith 2001). The somatotropic axis and especially IGFs, besides insulin, is involved in GIT development and especially in proliferation and maturation of enterocytes (Laburthe et al. 1988, Schober et al. 1990, Odle et al. 1996, MacDonald 1999, Menard et al. 1999). It has been demonstrated that IGF-II and insulin are involved in the mechanisms governing the differentiation of intestinal epithelium while IGF-I is mostly associated with crypt cell proliferation (Jehle et al. 1999). In newborn calves mRNAs for IGF-I and IGF-II, for receptors of growth hormone, IGF-I (IGF-IR), IGF-II (IGF-IIR) and insulin (IR), and for IGF binding proteins 1–3 are found in the ileum (Pfaffl et al. 2002). Based on ligand binding assays, receptors for IGF-I, IGF-II and insulin are present in the GIT of neonatal calves (Baumrucker et al. 1994, Hammon & Blum 2002). Therefore, IGFs and insulin ingested especially with colostrum (Odle et al. 1996, Xu et al. 1994, Blum & Baumrucker 2002), IGFs produced in the GI wall, and IGFs and insulin circulating in blood plasma can affect GI morphology and function after binding to their respective mucosal receptors (Lund 1994, Fholenhag et al. 1997, Jehle et al. 1999, Simmons et al. 1999, Blum & Baumrucker 2002). The effects of IGFs and insulin depend, at least in part, on receptor number and affinity, i.e. intestinal IGF-IR, IGF-IIR and IR may be involved in GIT development in pre-term calves. Because calves are born relatively mature compared with other species such as rats, mice and humans, differences with other species with respect to receptor numbers and thus of GIT responses to ingested food components can be expected, i.e. specific studies on IGF-IR, IGF-IIR and IR in calves are justified and needed. This study was conducted to investigate binding capacity (Bmax) and mRNA levels of IGF-IR, IGF-IIR and IR Journal of Endocrinology (2003) 176, 121–132

in the mucosa of duodenum, jejunum, ileum and colon in calves born 14 days pre-term and in calves born at normal term (after 290 days of pregnancy), in full-term calves on day 8 of life after being fed for 7 days, and in full-term 8-day-old calves fed different amounts of colostrum or only a formula (and thus different amounts of bioactive substances such as IGFs and insulin) for 7 days. We have tested the following hypotheses: (1) that mRNA levels and Bmax of IGF-IR, IGF-IIR and IR are specifically regulated and associated; (2) that IGF-IR, IGF-IIR and IR concentrations at the mRNA and protein level immediately after birth in pre-term calves differ from those of calves born at full-term; (3) that there are variations in IGF-IR, IGF-IIR and IR at the mRNA level in pre-term and full-term neonatal calves at different intestinal sites; (4) that IGF-IR, IGF-IIR and IR at the protein and mRNA level change within the first days of life in full-term neonatal calves; (5) that differences in intake of bioactive substances such as IGFs and insulin (as a consequence of differences in amounts of ingested colostrum or by ingesting only a formula) modify IGF-IR, IGF-IIR and IR mRNA levels and Bmax of duodenum, jejunum, ileum and colon; and (6) that IGF-IR, IGF-IIR and IR correlate with histomorphometrical traits and with proliferation rates of intestinal epithelium of duodenum, jejunum, ileum and colon. Materials and Methods Animals, husbandry, feeding and experimental design The experiments were performed according to the Swiss law on Animal Protection and the experimental procedures were approved by the Committee for Animal Experimentation of the Canton of Freiburg (GrangesPaccot, Switzerland), supervised by the Swiss Federal Veterinary Administration (Berne, Switzerland). Experiment 1 Single-born calves (n=19; seven Red Holstein, six SimmenthalRed Holstein, five Holstein Friesian and one Brown Swiss), originating from the Research Station, were assigned to three groups. Pre-term calves, born on day 277 of gestation after cows were injected with 500 µg prostaglandin F2 (Estrumate; Essex Pharma GmbH, Friesoythe, Germany) and 5 mg Flumethason (Flumilar; Veterinaria AG, Zürich, Switzerland), were killed immediately after birth before being fed, i.e. on day 1 of life (GrP, n=6). Full-term calves born after the normal duration of pregnancy (2902 days) were either killed immediately after birth (GrN, n=6) or on day 8 of life (GrC1–3, n=7). Calves of the GrC1–3 group were held on straw in individual pens for 7 days. Colostrum for GrC1–3 calves was from cows from the Research Station milked twice daily and was separately stored at – 20 C to make pools of milkings 1 to 6 for days 1, 2 and 3. Before feeding, colostrum was warmed to 40 C and then immediately www.endocrinology.org

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Table 1 Feeding plan for neonatal calves Group Fed amounts (g/kg BW/day)

GrCmax

GrC1–3

GrF1–3

Age (days) 1

60

Colostrum 1st milking

Formula 1

2

80


3

100


4

100

5

100

6

100

7

100

Colostrum 1st milking +MR (v/v=3:1) Colostrum 1st milking +MR (v/v=1:1) Colostrum 1st milking +MR (v/v=1:3) Colostrum 1st milking +MR (v/v=1:3)

Colostrum 1st +2nd milking Colostrum 3rd +4th milking Colostrum 5th +6th milking MR

Formula 2 Formula 3 MR

MR

MR

MR

MR

MR

MR

Calves of GrCmax were fed first-colostrum up to day 7 of life, calves of GrC1–3 were fed colostrum from milkings 1 to 6 during the first 3 days of life and then a milk replacer (MR; whose nutrient composition was similar to that of mature milk) and calves of GrF1–3 were fed a milk-based formula during the first 3 days of life and then a milk replacer. Calves were fed twice daily. The formula fed on days 1, 2 and 3 was milk-based and contained comparable amounts of nutrients as colostrum of day 1 (milking 1), day 2 (milking 3) and day 3 (milking 5) respectively, but contained only traces of growth factors and hormones such as IGF-I and insulin.

fed. From day 4 onwards calves were fed a milk replacer (MR) diluted with water (100 g/l water) up to day 7, mimicking usual husbandry conditions. The contents of colostrum from milkings 1 to 6 and the MR, together with the feeding plans have been published elsewhere (Blättler et al. 2001, Rauprich et al. 2000a,b). Calves were fed twice daily by bottle, beginning 2·70·7 h after birth. The ensuing feedings were at 8, 24, 32, 48, 56, and 72 h after the first feeding. From day 4 to day 7 calves were fed daily at 0800 and 1600 h. Starting on day 4 calves had free access to water. The MR (UFA-200-Natura; without antibiotics) was purchased from UFA AG (Sursee, Switzerland). Experiment 2 Male calves (n=21; 13 SimmentalRed Holstein, four Brown Swiss and four Holstein Fresian) were raised at the Swiss Federal Station for Animal Production (Posieux, Switzerland). All calves were single born after a normal length of pregnancy (290 days) and normal parturition. They were obtained immediately after birth and held on straw litter in individual pens up to day 7 of life. Calves were divided into three dietary groups (GrCmax, GrC1–3 and GrF1–3), each consisting of seven animals. Calves of the GrCmax group were fed twice daily during the first 3 days of life on pooled undiluted colostrum that was the first milking after parturition, and then on first-milked colostrum on days 4, 5, 6, and 7 that was diluted with 25, 50, 75, and 75 parts of MR respectively www.endocrinology.org

(Table 1). Calves of the GrC1–3 group received colostrum of milkings 1 to 6 (first 3 days of lactation) on the first 3 days of life and then MR (100 g/l water) up to day 7. Calves of the GrF1–3 group were fed a milk-based formula during the first 3 days of life and MR (100 g/l water) from day 4 to day 7. Calves were fed by plastic bottle twice daily. The total amounts of fed colostrum, formula and MR on days 1, 2, and 3–7 were 60 ml/kg body weight (BW), 80 ml/kg BW, and 100 ml/kg BW respectively. The first feeding was 2–4 h after birth, and then at 8, 24, 32, 48, 56, and 72 h after the first feeding. Starting on day 4 calves were fed daily at 0800 and 1600 h and had free access to water. Colostrum was collected from cows from the Research Station. Colostrum for GrCmax calves was a pool of first milking after parturition. Colostrum for GrC1–3 calves was obtained before the start of the experiment from cows milked twice daily. Colostrum of milkings 1 to 6 was used to prepare individual pools which were separately stored in plastic bottles at – 20 C. Before feeding, the colostrum was warmed to 40 C and immediately fed. Calves of the GrF1–3 group were fed three different milk-based formulas during the first 3 days of life (for meals on days 1, 2, and 3 respectively), containing only traces of growth factors and hormones such as IGF-I, IGF-II and insulin, but had comparable amounts of nutrients as colostrum on day 1 (from milkings 1 and 2), day 2 (from milkings 3 and 4) and day 3 (from milkings 5 and 6). Milk formulas were produced in co-operation with UFA AG. They consisted Journal of Endocrinology (2003) 176, 121–132

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Table 2 Composition of colostrum milkings, formula and milk replacer fed to calves during the first 7 days of life Day 1 Colostrum Parameters Dry matter (g/kg) Gross energy (MJ/kg) Crude protein (g/kg) Crude fat (g/kg) Nitrogen-free extracts (g/kg) Crude ashes (g/kg) IGF-I (µg/kg) Insulin (µg/kg)

215 5·4 109 60 33 14 292 20·0

Day 2 1

Formula

210 5·4 123 56 22 12 8 2·7

2

Day 3

Days 4–7

Colostrum

Formula

Colostrum

Formula

Milk replacer3

158 3·9 58 48 43 9 103 2·6

130 3·2 53 37 33 5 16 1·1

154 3·8 49 50 47 9 71 1·9

139 3·6 46 49 41 4 11 1·7

95 1·7 21 22 454 7 5 0·2

1

Colostrum pools were prepared from 8 cows and the data are the sums of milkings 1 and 2, 3 and 4, and 5 and 6 on days 1, 2 and 3 respectively. The formula contained (per kg) calcium caseinate (86, 208 and 282 g on days 1, 2 and 3 respectively), lactalbumin (576, 245 and 80 g on days 1, 2 and 3 respectively), milk fat (267, 262 and 353 g double cream on days 1, 2 and 3 respectively), lactose (57, 271 and 271 g on days 1, 2 and 3 respectively), and a mineral premix (14 g on days 1, 2 and 3 respectively). The mineral prefix (per kg) contained calcium (186 g), magnesium (224 g), sodium (31 g), phosphorus (92 g), chlorine (48 g), iron (12 g), manganese (8·1 mg), copper (1·6 mg), zinc (7·8 mg), iodine (0·03 mg), cobalt (0·02 mg) and selenium (0·02 mg). 3 The milk replacer contained (per kg) skim milk powder (550 g), whey (40 g), corn-derived products (dextrose, glucose, oat cream, starch; 172 g), tallow (145 g), lard (44 g/kg), lecithin (as emulgator; 19 g), calcium (12 g), phosphorus (7·5 g), magnesium (1·6 g), sodium (4·9 g), zinc (80 mg), manganese (60 mg), iron (20 mg), copper (8 mg), iodine (3 mg), selenium (0·5 mg), cobalt (0·5 mg), vitamin A (26·25 µmol retinol equivalent/kg), cholecalciferol (195 nmol/kg), vitamin E (360 µmol -tocopherol/kg), thiamine (57 µmol/kg), riboflavin (21 µmol/kg), vitamin B-6 (59 µmol/kg) and vitamin B-12 (37 nmol/kg). 4 Nitrogen-free extracts in milk replacer contained (per kg) 710 g lactose (i.e. 336 g lactose/kg milk replacer on a dry matter basis). 2

of calcium caseinate (a gift from Emmi Milch AG, Lucerne, Switzerland), lactoalbumin (a gift from Emmi Milch AG), lactose (UFA AG), milk fat in the form of commercially available dairy double cream provided by the Agricultural Institute of the Canton of Fribourg, Grangeneuve/Posieux, Switzerland, and a vitamin and mineral premix (Provini S.A., Cossonay-Gare, Switzerland). The milk replacer (UFA-200 Natura; containing no antibiotics) was purchased from UFA AG and was prepared as a 100 g/l solution. Composition of colostrum milkings, formula and milk replacer are presented in Table 2. To protect against infections calves were injected s.c. with 20 ml of an immunoglobulin (IgG) formulation (Gammaserin, 100 g IgG/l; E Gräub AG, Switzerland). In addition, from day 2 of life they received chicken eggderived immunoglobulin containing high titers against rotavirus and pathogenic E. coli type K99 (Lohmann Animal Health, Cuxhaven, Germany) with each meal. The amounts per meal were as follows: 5 g on day 2, 4 g on day 3, 3 g on day 4, 2 g on day 5, and 1 g on days 6 and 7. Intestinal tissue sampling Calves were slaughtered on day 8 of life or directly after birth (GrN and GrP), the abdominal cavity was opened and the GIT was immediately removed. Sections with a length of 15–20 cm that originated from the middle parts of different regions of the intestine - duodenum, jejunum, ileum and colon - were opened and washed with ice-cold saline. The mucosa was gently scraped, and was put into ice-cold buffer (50 mM Tris–HCl; 6 mM MgCl2, 1 mM Journal of Endocrinology (2003) 176, 121–132

EGTA; pH=7·4) for radioligand binding assays, or into TRIzol Reagent (Gibco BRL, Basle, Switzerland) before freezing in liquid nitrogen, and was stored at – 80 C until analyzed for mRNA of IGF-IR, IGF-IIR and IR. Radioligand binding assays The materials used and the binding assay procedures have recently been described in detail (Hammon & Blum 2002). Immediately after slaughter, the intestinal mucosa was homogenized using an Ultra-Turrax homogenizer (T25, Janke and Kunkel, Staufen, Germany) four times at low speed (8000 revs/min). The homogenate was then centrifuged at 800 g for 10 min, the supernatant was centrifuged at 10 000 g for 10 min, and then at 100 000 g for 1 h as described. The pellet was suspended in ice-cold buffer with a motor-driven Glass-Teflon homogenizer and the obtained membrane suspension was stored at – 80 C until analyzed. For binding studies the protein concentration of mucosal membrane suspensions was measured using a kit (BCA Protein Assay Reagent; Pierce, Rockford, IL, USA). The linearity of protein concentration was tested for each receptor at different protein concentrations. The final protein concentration of the membrane suspension was adjusted to 100, 50 and 200 µg protein/ml for [125I]IGF-I, [125I]IGF-II, and [125I]insulin binding studies respectively, to perform binding studies in a linear range. Receptors were defined based on differences in ligand binding affinities in competitive binding assays and based on ligand blots. For the quantification of binding of [125-I]IGF–I, [125-I]IGF–II and [125-I]insulin the radiolabeled ligand (0·35 ng) was incubated with increasing concentrations of the unlabeled ligand IGF-I www.endocrinology.org


(1012 to 107 M), IGF-II (1011 to 107 M), and insulin (1012 to 106 M) respectively, and Bmax and binding affinity (evaluated by determination of the 50% inhibition (IC50) of binding of the radioactive label by the unlabeled ligand) was calculated. In contrast to the previous study (Hammon & Blum 2002), IR were best fitted to a model with one binding site. The human/bovine IGF-I was donated by Novartis AG (formerly Ciba Geigy AG), St Aubin, Switzerland, IGF-II was purchased from GroPep, Adelaide, Australia, and bovine insulin was purchased from Sigma, St Louis, MO, USA. IGF-I, IGF-II and insulin were iodinated with the Chloramine T method. Determination of receptor mRNAs Total RNA extraction from mucosa of duodenum, jejunum, ileum and colon was performed using TRIzol Reagent (Gibco BRL) and was resuspended in RNasefree water treated with diethyl pyrocarbonate (DEPC, Sigma-Aldrich Vertriebs GmbH, Deisenhofen, Germany). RNA integrity and purity were tested by measurement of optical density and by electrophoresis using ethidium bromide staining. Total RNA was then reverse transcribed into cDNA using random hexamer primers (AmershamPharmacia Biotech) as described (Pfaffl et al. 2002). Materials used and the procedures followed in RT-PCR assays were recently described in detail (Pfaffl et al. 2002). The primers for IGF-IR (forward primer: TTA AAA TGG CCA GAA CCT GAG; reverse primer: ATT ATA ACC AAG CCT CCC AC), IGF-IIR (forward primer: TAC AAC TTC CGG TGG TAC ACC A; reverse primer: CAT GGC ATA CCA GTT TCC TCC A) and IR (forward primer: TCC TCA AGG AGC TGG AGG AGT; reverse primer: GCT GCT GTC ACA TTC CCC A) were generated by Microsyth GmbH (Balgach, Switzerland). RT-PCR quantification was performed with the LightCycle System (Roche Molecular Biochemicals, Rotkreuz, Switzerland) using software package 3·3 (Roche Molecular Biochemicals). Absolute quantification was based on external recombinant DNA standards (Pfaffl et al. 2002) and values were expressed on a molar basis. Histomorphometrical analyses and cell proliferation of intestinal epithelia For the histomorphometrical analyses hematoxylin and eosin-stained slides were available. Analyses were conducted with a light microscope connected to a videobased, computer-linked system, as described by Bühler et al. (1998) and Blättler et al. (2001). Cell proliferation was based on counting cells which incorporate 5-bromo-2 -deoxyuridine that was intravenously injected 1 h before euthanasia (Blättler et al. www.endocrinology.org

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2001). 5-Bromo-2 -deoxyuridine-labeled (mitotic) crypt cells were calculated relative to unlabeled crypt cells and served as an indicator of cell proliferation rate. Data evaluation and statistics For data analysis, the IC50 of radiolabeled binding by the unlabeled ligand and Bmax were calculated by weighted least squares curve fitting using the GraphPad computer program (GraphPad Software Inc., San Diego, CA, USA). The results from real-time RT-PCR quantification on Light Cycler are expressed in fmol mRNAs per 1 mg total RNA. The Bmax, IC50, mRNAs values, and Bmax/ mRNA ratios are given as means S.E.M. Group differences of Bmax, IC50, and mRNAs were evaluated using the RANDOM and REPEATED methods of the MIXED procedure with an inter-animal random effect of differences between the animals and a correlation structure within animals (SAS 1995). Age (experiment 1), feeding (experiment 2), and gut segments were used as fixed effects within animals. Differences (Pduodenum and ileum in GrN) and between groups (GrN>GrP in colon; GrN>GrC1–3 in jejunum, colon and total intestine). Although total RNA concentration was higher in GrC1–3 than in GrN, IGF-IIR mRNA concentrations per g wet tissue were still significantly higher in GrN than in GrC1–3 (data not shown). Expression of IR differed significantly between intestinal sites (in GrP and GrN it was lowest in ileum; in GrC1–3 it was highest in colon), but there were no significant group differences. Bmax of IGF-IR (Table 4) differed significantly between intestinal sites (ileum and colon>duodenum and jejunum Journal of Endocrinology (2003) 176, 121–132

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Table 3 Concentrations of mRNA (fmol/mg total RNA) for IGF-I, IGF-II and insulin receptors in intestinal mucosa of pre-term calves (GrP), full-term calves (GrN), and 8-day-old calves (GrC1–3) (experiment 1). Results are expressed as means and pooled

Group differences (P values)

Group S.E.M.

GrP vs GrN

GrC1–3 vs GrN

Pooled GrP IGF-IR Duodenum Jejunum Ileum Colon Total intestine IGF-IIR Duodenum Jejunum Ileum Colon Total intestine IR Duodenum Jejunum Ileum Colon Total intestine

S.E.M.

GrN

GrC1–3

3·25 2·56 1·88 2·66 2·59

2·96 2·02 2·07 3·12 2·55

2·15A 0·84B 1·56AB 1·69AB 1·53

0·76 0·53 0·55 0·63 0·30

NS NS NS NS NS

NS NS NS NS NS

0·085 0·077 0·05 0·072 0·071

0·063B 0·1AB 0·074B 0·15A 0·1

0·022 0·035 0·028 0·027 0·028

0·013 0·02 0·01 0·03 0·009

NS NS NS 0·01 NS

NS 0·05 NS 0·001 0·01

3·3 3·5 1·8 4·5 1·8

NS NS NS NS NS

NS NS NS NS NS

16·9A 15·3A 7·2B 14·6A 13·5

11·2AB 12·8A 5·8B 14·2A 11·5

9·6AB 5·8B 8·2B 15·3A 9·7

Calves of GrP (n=6) were born 2 weeks pre-term (after 277 days of pregnancy) and those of GrN (n=6) were born full-term (after 290 days of pregnancy) and were killed immediately after birth. Calves of GrC1–3 (n=7) were born full-term, fed colostrum derived from milkings 1 to 6 for 3 days and then milk replacer (whose nutrient content was similar to that of mature milk) up to day 7 and were killed on day 8. A,B Within a column means of IGF-IR, IGF-IIR and IR with different capital superscript letters are significantly different (P0·05).

in GrP, ileum and colon>jejunum in GrN; colon> jejunum in GrC1–3) and between groups (GrP>GrN in colon). Bmax of IGF-IIR differed significantly between intestinal sites (duodenum and colon>jejunum and ileum in GrP; duodenum>ileum and colon>jejunum in GrN; duodenum, jejunum, and colon>ileum in GrC1–3) and between groups (GrN>GrP in duodenum, ileum, and total intestine; GrN>GrC1–3 in duodenum, ileum, colon, and total intestine) and there was also a significant groupgut interaction. Bmax of IR differed significantly between intestinal sites (ileum>duodenum, jejunum and colon in GrC1–3) and between groups (GrN>GrP in total intestine; GrC1–3 >GrN in ileum and total intestine) and there was a significant groupgut interaction. Experiment 2: concentrations of mRNA and maximal binding capacities of IGF-IR, IGF-IIR and IR in the mucosa of duodenum, jejunum, ileum and colon of 8-day-old calves fed different amounts of colostrum or a milk-based formula Expression of IGF-IR (Table 5) was significantly different between intestinal sites (ileum>colon, duodenumd jejunum in GrF1–3) and there were significant group Journal of Endocrinology (2003) 176, 121–132

differences (GrF1–3 >GrCmax and GrC1–3 in ileum). In addition, there was a significant groupgut interaction. Expression of IGF-IIR in intestinal mucosa did not differ significantly between different intestinal segments, but IGF-IIR expression in duodenum tended to be higher (Pduodenum, jejunum, ileum in GrCmax; colon>jejunum and ileum in GrC1–3) and between groups (GrCmax >GrF1–3 in colon). There was also a significant groupgut segment interaction. Bmax of IGF-IR (Table 6) was significantly different between intestinal sites (colon>jejunum in GrC1–3 and duodenum, ileum, and colon>jejunum in GrF1–3) and between groups (GrCmax >GrF1–3 in jejunum). There was a significant groupgut interaction. Bmax of IGF-IIR was significantly different between intestinal sites (duodenum, jejunum and colon>ileum in all three groups), but there were no significant group differences. Bmax of IR was significantly different between intestinal sites (ileum>duodenum, jejunum and colon in all three groups) and tended to be different (though not significantly, PGrF1–3 in jejunum and total intestine; GrCmax >GrC1–3 in colon). www.endocrinology.org


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Table 4 Binding capacities (Bmax; fmol/mg protein) of IGF-I, IGF-II and insulin receptors in intestinal mucosa of pre-term calves (GrP), full-term calves (GrN), and 8-day-old calves (GrC1–3) (experiment 1). Results are expressed as means and pooled S.E.M. Group differences (P values)

Group Pooled GrP IGF-IR Duodenum Jejunum Ileum Colon Total intestine IGF-IIR Duodenum Jejunum Ileum Colon Total intestine IR Duodenum Jejunum Ileum Colon Total intestine

7·2B 6·3B 12·2A 15·3A 10·2 176A 60B 66B 166A 119 1·7 0·9 2·4 3·0 1·9

GrN

7·9AB 5·1B 11·3A 9·8A 8·5 232A 76C 139B 160B 151 3·6 2·4 5·8 3·8 3·8

GrC1–3 9·3AB 7·6B 10·7AB 12·0A 9·7 56A 63A 19B 63A 51 5·0B 4·1B 21·6A 3·1B 8·5

S.E.M.

1·28 1·11 1·89 2·38 0·98 21·3 12·7 11·7 21·3 12·9 0·53 0·57 1·80 0·61 0·85

GrP vs GrN

GrC1–3 vs GrN

NS NS NS 0·05 NS

NS NS NS NS NS

0·05 NS 0·01 NS 0·05

0·001 NS 0·001 0·001 0·001

NS NS NS NS 0·05

NS NS 0·001 NS 0·001

A,B,C Within a column means of IGF-IR, IGF-IIR and IR with different capital superscript letters are significantly different (P0·05). For further details see footnote to Table 3.

Comparisons between IGF-I, IGF-II and insulin receptors and relationships between intestinal mucosal mRNAs and binding capacities of IGF-I, IGF-II and insulin receptors

Correlations between IGF-I, IGF-II and insulin binding capacities with intestinal epithelial histomorphometric and proliferative measurements

In this section analyses were performed based on pooled data of all groups and all intestinal sites of both experiments. Concentrations of IR mRNA were the highest (P